In recent years a number of nucleic acid molecules, including synthetic oligonucleotides, have been described as immunostimulatory molecules. These immunostimulatory nucleic acid molecules include CpG-containing oligodeoxynucleotides (ODN), which signal through Toll-like receptor 9 (TLR9), as well as double-stranded RNA (dsRNA), which signals through Toll-like receptor 3 (TLR3), and single-stranded RNA and oligoribonucleotides (ORN), which signal through Toll-like receptors 7 and 8 (TLR7 and TLR8).
Very potent induction of interferon by short single-stranded RNAs (ssRNAs), which were obtained by in vitro transcription with T3, T7 and Sp6 RNA polymerases, has also been reported. Kim D H et al. (2004) Nat Biotechnol 22:321-5. This report further disclosed that a 5′-triphosphate on the in vitro-transcribed ORN is required for interferon (IFN) induction. These findings were also disclosed in U.S. Patent Application Publication US 2006/0178334 A1.
It was separately reported that 5′-triphosphate RNA is the ligand for retinoic-acid-inducible protein I (RIG-I) and that the 5′-triphosphate group serves as molecular signature for the detection of viral RNA leading to strong IFN induction. Hornung V et al. (2006) Science 314:994-7.
5′-Triphosphate groups are energetically activated compounds with a propensity to self-decompose by hydrolysis. Under in vivo conditions, triphosphates are metabolically cleaved, e.g. by 5′-phosphatases such as calf intestinal phosphatase (CIP), and RNA is capped, such that most, if not all, cytoplasmic RNA lacks exposed 5′-triphosphates.